title: Genetic construction, properties and application of a green fluorescent protein-tagged ciliary neurotrophic factor.
abstract: The in vitro and in vivo actions of ciliary neurotrophic factor (CNTF) suggest that endogenous CNTF plays a role in nervous system development and maintenance. CNTF produces most, possibly all, of its effects by binding to a protein referred to as CNTF receptor alpha (CNTFRalpha). Information on CNTFRalpha tissue expression and dynamics would be advanced by the availability of reagents suitable for studying the subcellular localization and trafficking of CNTFRalpha. This paper describes the genetic construction, synthesis, purification and properties of a chimeric protein in which a highly fluorescent form of the green fluorescent protein (GFP) has been fused to human CNTF. The fusion protein, termed GFP-CNTF, was expressed in Escherichia coli. Histidine tagging of GFP-CNTF permitted ready purification by means of immobilized Ni(II) chromatography. Under non-reducing conditions GFP-CNTF migrated on SDS-PAGE with an apparent molecular mass of 50 kDa, although under reducing conditions it behaved electrophoretically as a 67 kDa species. Despite these discrepancies, the molecular mass of GFP-CNTF determined by mass spectrometry (54755) agreed well with its deduced relative molecular mass of 54536. Importantly, the absorbance profile of the GFP chromophore in GFP-CNTF was not modified by the presence of the CNTF domain. Moreover, the fluorescence emission spectrum of GFP-CNTF overlapped that of GFP, showing neither a change in absorbance shift nor a difference in the fluorescence quantum yield. Circular dichroism spectroscopy confirmed that the CNTF and GFP domains of GFP-CNTF folded independently of each other. GFP-tagged CNTF was equipotent to human CNTF in supporting the survival of cultured embryonic chicken sensory and ciliary ganglion neurons. GFP-CNTF, but not GFP, bound to immobilized CNTFRalpha and was displaced by an excess of human CNTF. GFP-CNTF specifically labeled the Purkinje cell layer in cerebellar slices from adult rat. This report is the first to describe a GFP chimera with a neurotrophic factor as the fusion partner. GFP-CNTF should provide a valuable tool for elucidating the role of CNTFRalpha in nervous system function.
authors.affiliation: Department of Biological Chemistry, CRIBI, University of Padova, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Negro
authors.forename: L
authors.initials: L
authors.lastname: Grassato
authors.forename: P
authors.initials: P
authors.lastname: Polverino De Laureto
authors.forename: S D
authors.initials: SD
authors.lastname: Skaper
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020934:Ciliary Neurotrophic Factor; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011689:Purkinje Cells / Q000378:metabolism; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D020801:Receptor, Ciliary Neurotrophic Factor; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013054:Spectrophotometry, Atomic
chemical_list: D020934:Ciliary Neurotrophic Factor; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D020801:Receptor, Ciliary Neurotrophic Factor; D017475:Receptors, Nerve Growth Factor; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D020794:Receptor Protein-Tyrosine Kinases